Chemical sensor arrays can be used to identify and classify complex gas mixtures or odors (Shurmer, H. V., An electronic nose: A sensitive and discriminating substitute for a mammalian olfactory system, IEEE proc. G 137, 197-204, 1990; Gardner, J. W. and Bartlett, P. N. (eds), Sensors and Sensory Systems for an Electronic Nose, Proc. NATO Advances Research Workshop, Reykjavik, 1992.) Chemical sensors are in general non-specific, but have different selectivity patterns towards the species in the odor. More specifically, it has been demonstrated how large sensing surfaces consisting of different catalytic metals in metal-oxide-semiconductor field effect structures can be used together with an optical evaluation technique to obtain visually identifiable images of odors (I. Lundström, R. Erlandsson, U. Frykman, E. Hedborg, A. Spetz, H. Sundgren, S. Welin, and F. Winquist, Artificial ‘olfactory’ images from a chemical sensor using a light-pulse technique Nature, 352, 47-50, 1991. It is important to note that this increased informational content is derived from the (continuous) varying selectivity profile along the sensing surface for the sensor array. No discrete recognition elements are known to exist. Different pattern recognition methods based on statistical approaches or artificial neural networks can be used to evaluate the signal patterns from these sensors. The devices have been used to analyze a variety of food stuffs (Winquist, F., Hörnsten, E. G., Sundgren, H. and Lundström, I., Performance of an electronic nose for quality estimation of ground meat, Meas. Sci. Technol. 4,1493-1500, 1993; Winquist, F., Hörnsten, G., Holmberg, M., Nilsson, L. And Lundström, I. Classification of bacteria using a simplified sensor array and neural nets”, submitted).
New sensor concept. The analogy between these sensors and that of biological sensing systems, such as the olfactory system, has been conceptually important in driving the development of this technology. The basis for the human olfactory sense is that a signal pattern is generated from the receptors cells in the olfactory bulb. The receptor cells are not specific for a particular molecules, but rather belong to different selectivity classes. The basis for olfaction (smell) appears to combine the signals obtained from each of the low specificity receptor classes. The combinatorial effect that results leads to an increase in the discriminatory ability of the system (despite the relatively small number of receptor classes). The chemical sensing elements can recognize odors but lack the discrete recognition capabilities that biomolecules and synthetic biomimetic molecules possess. As noted, chemical sensors use continuous gradients and other approaches as recognition elements and are not discrete. Nature uses discrete identifiable sensing elements which have evolved recognition capabilities in a biological context. One object of the invention is to apply discrete biosensing elements in a fashion that increases the informational content of the diagnostic assay. This would require the employment of a biomolecule with broad recognition characteristics which would normally be considered too ill-defined to be useful in conventional diagnostics. The specificity must be chosen so as to obtain adequately broad binding (high informational content) but not so much as to make differentiation between specific and nonspecific binding impossible, i.e. adequate signal to noise ratio. At the same time, biological sensing elements must have well defined binding characteristics that are appropriate for this assay strategy.
The invention described here involves the development of a new assay strategy for complex sample discrimination using arrays of biorecognition elements that is far more informationally rich than conventional assays. Another object of this invention is to reduce the number of tests that must be performed before a diagnosis can be made, thereby reducing the time required to start treatment as well as the cost. Unlike standard diagnostic tests which detect known compounds highly specifically, we detect the binding of unknown compounds to the lectins. Thus, the new assay strategy requires the employment of specialized nonlabel-based detection techniques, including but not limited to quartz crystal microbalances and optical techniques such as optoaucostics, reflectometry, ellipsometry and surface plasmon resonance (SPR). All of the methods that are based on polarized light reflected off a solid surface have already proven valuable for thickness determination of proteins on solid surfaces. The sensitivity of the methods are about the same, which is on the order of a few angstroms.
Biological sensing elements. Proteins have the ability to combine specifically and reversibly with a variety of ligands. Enzymes for example bind substrates and inhibitors while mL, antibodies can be produced which bind a variety of antigens such as carbohydrates, proteins, and small molecules. Another class of proteins, lectins, have the ability to bind sugars and are devoid of enzymatic activity. Receptors bind a wide range of ligands with high affinity and specificity. Nature evolves and maintains proteins for specific purposes with adequate affinity and specificity for a particular purpose. Thus, the employment of biological or synthetic biomimetic sensing elements is the most appropriate approach for identifying changes that are of biological significance. We have chosen to test the biosensing affinity arrays invention described here using the lectins. We shall describe lectins and give several advantages this class of proteins has over the more commonly used immune-based diagnostics in the application of this invention.
Lectins as biological recognition elements. As mentioned previously, lectins bind carbohydrates and to compounds with similar structure. (Lectins as molecules and as tools. Lis, H. And Sharon, N. Ann. Rev. Biochem., 55, 35-67, 1986; Advances in Lectin Research. Vol 1, Franz, H. Ed., Springer-Verlag, Berlin, 187 pp., 1987). Lectins also have the capability to agglutinate cells, precipitating polysaccharides and glycoproteins and are of nonimmune origin. This is due to the fact that they are oligomeric in structure, usually containing one sugar binding site per subunit. In this respect, lectins have agglutinating abilities similar to those of antibodies. They also can be inhibited by low molecular weight compounds, which in the case of lectins are small carbohydrates, such as monosaccharide, oligosaccharides or macromolecules which contain them.
First, lectins provide a broad spectrum of well defined binding specificities with a high degree of cross reactivities as compared for example with antibodies and enzymes. Furthermore, they are stable and have a wide range of affinities and specificities. In addition, over one hundred lectins have been characterized. It now appears that lectins mediate a variety of cellular interactions during development and in the adult animal (Drickamer, K. And Taylor, M. E. Biology of Animal Lectins Annu. Rev. Cell Biol. 9:237-64, 1993). This is supported by data which shows that lectin expression patterns change throughout development and in response to a wide range of environmental changes [Varki, A. Biological roles of oligosaccharides: all of the theories are correct, Glycobiology, 3:97-130, 1993.]. The involvement of oligosaccharides in selectin-mediated cell—cell recognition by the immune system in response to inflammation [Lasky, L. A. Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science 258:964-969, 1992 and sperm-cell recognition during fertilization [Miller, D. J., Macek, M. B., Shur, B. D. Complementarity between sperm surface b1,4-galactosyltransferase and egg coat ZP3 mediates sperm-egg binding. Nature 357:589-593, 1992) are but a few examples. It is also known that modifying the expression of glycosides and glycosyltransferases interferes with normal development. However, it has not been possible to define the individual contributions of individual monosaccharides residues and oligosaccharide chains to stage-specific and tissue-specific developmental processes.
An object of this invention is the ability of the assay strategy to discriminate complex samples which could be used to delineate complex basic developmental processes.
Second, the study of lectins is intimately linked to that of carbohydrates and is referred to as glycobiology (Glycoproteins, Hughes, R. C. outline Studies in Biology, Chapman and Hall, London and New York, 95 pp, 1983). Glycosylation is used extensively in nature for a wide range of purposes some general, such as protease protection, some directed to particular classes of proteins, such as signaling mechanism for clearance of proteins from serum and some highly specific, such as cell adhesion. Carbohydrates also act as control mechanisms, as signals for cellular localization, for specific cell surface recognition of one cell type by another, for clearance of a particular glycoform from serum, assist in protein folding possibly by providing protection against proteolysis (Pareth, R. B. Effects of glycosylation on protein function. Curr. Opin. Struct. Biol. 1:750-54, 1991).
Carbohydrates contain a potential informational content several orders of magnitude greater than any other biological oligomer. For example, if one calculates the number of possible structures for a hexamer of sugars and that of a hexamer of amino acids, the figure is >1.05×1012 and 4.6×104. The difference is more than seven orders of magnitude. Accordingly, sugars clearly provide the largest single source of diversity in the biological world (Laine, R. A. Invited Commentary in Glyco-Forum section Glycobiology 1994 8, 759-767).
Lectins have also been shown to be important in defence against a variety of pathogens. The mannose binding lectins in animals mediates antibody-independent binding of pathogens which contain a high concentration of mannose on their surface. These monosaccharides are not generally found in terminal positions on serum or cell surface glycoproteins in mammalian systems. The recognition event can initiate the complement cascade [Ikeda, K, Sannoh, T., Kawasaki, T. And Yamashima, I. (1987) J. Biol. Chem. 262, 7451-7454.]. Plant lectins have also been implicated in attachment of symbiotic nitrogen fixing bacteria to the roots of leguminous plants and int eh protection of plants against fungal pathogens (Bohlool, B. B. and Schmidt, E. L. (1974) Science 185:269-71).
Third, numerous pathogens use carbohydrate-lectin interactions in order to gain entry into their hosts. For example, bacteria and intestinal parasites, such as amoeba, mediate the sugar specific adherence of the organisms to epithelial cells and thus facilitate infection. (Liener, I. E., Sharon, N., Goldstein, I. J. eds (1986) The Lectins: Properties, functions and applications in biology and medicine. New York: Academic.). Viruses such as influenza virus (myxovirus) and Sendia virus (paramyxovirus) use a haemagglutonin protein that binds sialic acid containing receptors on the surface of target cells to initiate the virus-cell interaction (Paulsson, J. C. Interaction of animal viruses with cell surface receptors, in: The Receptors (Vol. 2) (ed. P. M. Conn), Academic Press, New York, pp. 131-219, 1985).
Another object of the invention is to study the pathogenesis of diseases that use carbohydrates or lectins in order to gain entry into cells.
Carbohydrate binding proteins such as selectins are believed to play a critical role in immune responses including inflammation (Springer, et al. 1991 Nature 349:196-197; Philips, et al., 1990 Science 250:1130-32. Specific carbohydrate ligands have been identified and have been used to control inflammation, immunosuppression, etc. through their interaction with selectin proteins and/or other lectins (Gaeta, et al., U.S. Pat. No. 5,576,305 corresponding to U.S. patent application Ser. No. 07/538,853, filed 15 Jun. 1990; Ippolito, et al., U.S. Pat. No. 5,374,655 corresponding to U. S. patent application Ser. No. 07/889,017, filed 26 May 1992). Other glycoproteins have also been shown to be useful in suppressing mammalian immune responses (Smith et al., U.S. Pat. No. 5,453,272 U.S. patent application Ser. No. 07/956,043 filed 2 Oct 1992).
Another object of the invention is to use the assay strategy in order to delineating the more subtle recognition functions of lectins, including but not limited to selectin and other lectins, in immune and inflammatory responses.
Fourth, the wide distribution of and ready availability of large numbers of sugars and sugar binding proteins combined with their ubiquity throughout nature, has led to their extensive use as reagents for studying carbohydrates in solution and on cell surfaces. They were originally used for blood typing (Lis and Sharon), for the identification and separation of cells (Sharon, N. 1983 Adv. Immunol. 34:213-98). Labelled lectins serve as specific reagents for the detection of glycoproteins separated on gels, either directly or after blotting (Rohringer, R., Holden, D. W. 1985 Anal. Biochem. 144:118-27.) Immobilized lectins are routinely used for isolating glycoproteins such as the insulin receptor (Hedo; J. A. Harrison, L. C. Roth, J. 1981 Biochemistry 20:3385-93) and the many others proteins. Lectins have been widely used to separate cells such as thymocytes and splenocytes (Reisner, Y, Sharon, N. 1984 Methods Enzymol. 108:168-79; Maekawa, M., Nishimune, Y. 1985 Biol. Reprod. 32:419-25.). Numerous bacteria have been typed using lectins (Doyle, R. J., Keller, K. F. 1984 Can. J. Microbiol. 3:4-9; DeLucca, A. J. II 1984 Can. J Microbiol. 3:1100-4). Primates can be differentiated from non-primates by the presence of specific sugar residues [Spiro, R. G. and Bhoyroo, V. D. (1984) J. Biol. Chem. 259, 9858-9866; Galili, U., Shohet, S. B., Kobrin, E. Kobrin, E., Stults, C. L. M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762. These applications are strictly dependent upon the ability of a particular lectin to specifically identify a carbohydrate attached either to a soluble biomolecule or to a cell or organelle.
Fifth, most cells have a coating of carbohydrate chains in the form of membrane glycoproteins and glycolipids (in eukaryotes) or of polysaccharides (in prokaryotes). In eukaryotes, the cell type and environmental factors such as glucose concentration, play a major role in determining the extent and type of glycosylation, which is both species and tissue specific (Parekh, R. B., Dwek, R. A., Thomas, J. R., Opdenakker, G., Rademacher, T. W. (1989) Biochemistry 28, 7644-7662; Goochee, C. F. and Monica, T. (1990) Bio/Technology 8, 421-427). In addition, each individual enzymatic reaction may or may not go to completion, giving rise to glycoforms or glycosylated variants of the protein (Rademacher, et al., Ann. Rev. Biochem., 1988 57:789-838). These factors give rise to the enormous heterogeneity of carbohydrate structures found in vivo that has hindered their analysis. However, in some instances the relative concentration of the different forms have been shown to vary in specific ways in certain health and disease states. For example This also explains why glycosylation patterns of natural glycoproteins may be influenced by physiological changes such as pregnancy and also diseases such as rheumatoid arthritis.
In addition, it is known that the interaction between individual monosaccharides and CRDs is too weak to account for the affinities that lectins have for glycoproteins. The oligomeric lectins (multivalent) clusters the carbohydrate recognition domains (CRDs) which increases both the specificity and the affinity for multibranched oligosaccharides. While these effects are not well understood, it is clear that the density of CRD has biological significance. Thus, is an additional parameter that can be used in the invention to further increase the informational content of the assay. This would indicate that lectins could be useful following changes in the overall state of complex biological samples. This wealth of diversity provides a nearly unlimited range of sensor elements from which to choose.
It is believed that the multivalency of lectins for carbohydrates is important for their biological activity. Thus, an object of the invention would be the application of density gradients of lectins on surfaces in continues and discontinuous, as well as in homogeneous and heterogeneous formats for sample discrimination. This would provide a unique tool for gaining a basic understanding of the effect of binding site density on the recognition process. Methods are available to those skilled in the art for adapting reflectometry, ellipsometry or SPR for scanning and imaging modes. This also would provide an additional assay parameter, thus increasing the informational content of the lectin affinity arrays and thereby improving their ability to discriminate complex samples.
Diagnostic assays strategies. Immunoassay based diagnostics currently predominate the market, nevertheless, lectins provide some advantages over conventional immunoassays. Lectins are present in most life forms and more importantly they are found in life forms such as plants, microorganisms and viruses, which do not synthesize immunoglobulin. Clearly the biological function(s) of lectins precedes that of the immune system, many of which are unknown at present. Thus, these sensing elements will be more useful for identification and classification purposes. The extensive homologies observed between different classes of lectins demonstrate that these proteins have been conserved throughout evolution and provide strong evidence that they have important function(s) in biology. Another difference is that lectins are structurally diverse whereas antibodies are structurally similar. This structural diversity would result in a corresponding diversity of stabilities that would increase the flexibility of the assay formats (antibodies tends to denature under similar conditions due to their structural similarity). Thus, lectins combine the multivalency of antibodies with the structural diversity of enzymes. Other proteins which bind carbohydrates also exist such as those that participate in carbohydrate metabolism and sugar transport. In general, these proteins only bind one carbohydrate and serve quite different purposes than lectins.
The detection of specified antigens, haptens and the like substances in bodily fluids such as blood, serum, sputum, urine, and the like is of central importance in both research and clinical environments. The detection of such ligands can often be correlated to various disease states and consequently, is of great importance in diagnosis and for gaining a basic understanding concerning the genesis of disease, as well as for monitoring the efficacy of therapeutic treatments. The large and ever increasing ability to diagnose and treat diseases has lead to an explosive increase in demand for diagnostic testing. And while the cost per assay has been reduced, the number of tests that are performed has increased dramatically. This is in part due to the increasing number of tests that are available and in part due to the need medical practitioners have to be able to justify their actions in the event that legal action (malpractice suits) should be taken against them.
Accordingly, improved methods for detecting ligands in aqueous samples are constantly being sought. In particular, such preferred methods or assays are those that are faster, more flexibility, simpler to perform and manufacture, as well as having low manufacturing costs. In addition, there is an increasing need for strategies that will reduce the time necessary to develop diagnostic assays for such agents as HIV and Bovine Spongiform Encephalitis (BSE). Increasing health costs require the development of new, rapid, and more effective diagnostic strategies.
In general, immunoassays are based upon the immunological reaction between proteins such as antibodies, antibody fragments, or even artificially generated elements simulating antibody binding sites such as peptides, templated polymers and the like (hereafter referred to as antibody recognition) and the substance for which they are specific, the ligand. Immunological reactions are characterized by their high specificity and accordingly, numerous schemes have been developed in order to take advantage of this characteristic. The goal is to identify a particular state with absolute specificity using as few assays as possible.
In the traditional heterogeneous forward assay, an antibody is immobilized on a solid phase such as microparticles, microtiter wells, paddles, and the like. The sample is then contacted with the immobilized antibody and the ligand binds if present in the sample. The bound substance is detected and quantitated by an entity associated directly or indirectly therewith. Such detectable entity include fluorescent molecules, chemiluminescent molecules, enzyme, isotopes, microparticles and the like. Many variants have been developed such as competition, indirect competition, and the like. Various methods are available to those skilled in the art for quantitating the amount of substance bound using these assays.
In addition to immunoassays, other diagnostic assays are available based upon the same demand for absolute specificity using wide range of recognition elements such as proteins (lectins, receptors, and the like), nucleic acids, carbohydrates, lipids and/or synthetic/engineered biomimetic compounds and the like. A wide range of basic techniques have also been developed including but not limited to microscopy, chromatography and electrophoresis in order to specifically identify diseases.
It is an object of this invention to provide an assay strategy for sample discrimination which relies upon an array of sensing elements with low specificity in order to increase the informational content of the diagnostic assay. The assay strategy is capable of discriminating subtle changes and thus allows early identification of changes in the state of health that can be of crucial importance. In some instances, the sensing elements used in conventional assays will be applicable. However, in most instances the specificity of these reagents will be too high to allow their use. Accordingly, new screening procedures will be developed in order to isolated reagents with appropriate combination of affinities and specificities and is an object of this invention, as well.
The assay strategy can be extended to a wide range of applications that require complex sample discrimination including but not limited to identification of diseases, identification of changes caused by the disease itself in the host (including but not limited to human, animal, plants and microorganisms). Complex samples containing biological material and/or degradation products including but not limited to such as food stuffs like beverages, dry foods, and the like (including but not limited to quality control, for detection of unwanted microbial growth, freshness, physical damage), as well as the control of environmental samples for microbial flora (including but not limited to microbial content and composition), pollutants and their breakdown products in air, soil and water samples. This strategy and assays based on it could also be used for monitoring fermentation processes, including but not limited to yogurt, beer, wine and the like, broths, as well as in fermentation processes in which products are produced such as biological compounds produced by microbial processes, such as insulin from genetically engineered bacteria and the like, as well as condiments made for seasoning and the like, as well fermentation processes used in the production of animal food stuffs.
Current diagnostic testing approaches used to determine the general state of health such as hemagloblin, blood pressure and the like give only limited information. And while these tests provide useful information as to the general state of health, they do not provide adequate information to identify diseases nor are they sensitive enough to detect subtle changes required for early disease detection. There is a need, therefore, to develop new strategies for the identification of disease states which provide information as to which class of ailments the patient is suffering in order to reduce the number of specific tests which must be performed.
Another object of the invention is to provide a strategy and assays for improved techniques to monitor the general state of health to assist efforts in identifying ailments early on thereby allowing treatment to begin at an earlier stage than would have been possible otherwise. The early treatment of disease has been shown to reduce health care costs. This strategy would also be useful in preventative health care schemes. A similar situation exists in food stuff and environmental testing.
This diagnostic strategy based on the use of discrete recognition elements with broad recognition specificities combined with computer based artificial neural network data analysis can also be used with discrete synthetic biomimetic recognition elements with appropriate specificity (signal to noise). These could be made from modified biological material or from polymeric materials by conventional templating techniques and the like. This embodiment of the invention would be especially useful in applications requiring assay conditions that would destroy or dramatically reduce the binding affinty and/or specificity of conventional biological sensing elements including but not limited to organic solvents, high or low temperature, acidic or basic solutions and salts.
These detection techniques demand highly reproducible, high density immobilization methods for flat surfaces such as silicon wafers or flat glass. Other surfaces compatible with these detection techniques including but not limited to plastic, silicon, mica and glass surfaces in both metal coated or uncoated. Standard immobilization protocols resulted in poor overall reproducibility due to inadequate signal to noise ratios. A method was developed that allowed high density immobilization of biomolecules with high retention of biological activity while minimizing nonspecific binding assay. The increased sensitivity and reduced nonspecific binding achieved increased the signal to noise ratio that was essential for this assay strategy. We now believe that the nearly 100% surface coverage. This prevents interaction directly with the metal surface, and provides an essentially homogenous interaction matrix, and maximizes surface densities.
The strategy could be used to discriminate complex samples from other origins including but not limited to, body fluids such as blood, serum, saliva, sputum, urine and the like., thus allowing complex correlations with known reference standards (using pattern recognition programs). Environmental samples such as air, soil, water and the like, food stuffs and the like as well as artificial substances for which appropriate sensing elements can be found could be analysed using this strategy, i.e. appropriate signal to noise ratios can be obtained for the samples in question. No analytical approach can currently exists which can discriminate samples as rapidly or as cost effectively. An important object of the invention is the ability of the strategy to take advantage of as yet unknown recognition functions present in the recognition elements.
We have not made any attempt to identify the substances bound to the lectin arrays but various methods are available to those skilled in the art of identifying biomolecules to perform this type of analysis. While this is not the primary aim of the invention, it may prove useful for understanding the nature of changes that have occurred that may assist in the development of therapies and/or the development of therapeutic drugs. In addition, any recognition element which exhibits the characteristics required by this assay strategy, including but not limited to biomolecules such as proteins, lipids, carbohydrates and nucleic acids, modified biomolecules, such as genetically engineered, chemically modified, and the like, as well as synthetic molecules used in molecular recognition, such as cyclodextrans, templated and imprinted polymers and the like, may also be used in this regime.
Another object of the invention is the combined approach used to immobilize the biomolecules and included special surfaces (gold), hydrophobic thick-film patterning, self-assembling long chain thiols with terminal carboxylic acid groups and an empirically determined EDC/NHS immobilization protocol. While all of these have been used individually, no immobilization protocol exists which combines these various techniques into a single unified protocol.
Numerous patents have been disclosed which employ a wide range of biological sensing elements for diagnostic and therapeutic purposes, such as WO 95/29692, WO 95/15175, WO 95/28962, WO 95/07462, Canadian patent 2,133,772, U.S. Pat. No. 4,289,747, U.S. Pat. No. 4,389,392, U.S. Pat. No. 4,298,689 and WO 95/26634. All of these inventions use the unique specificity of some sensing element, be it an antibody or a lectin, to identify a single disease (or groups of highly related diseases). Great attempts are made to increase the specific reaction and reduce the nonspecific reactions, in strong contrast to the invention described here.
WO patent 92/19975 describes a method for labelling glycoproteins with a fluorescent molecule in a complex mixture using a carbohydrate specific labelling reagent. This mixture of labelled proteins is separated and the banding pattern analysed using pattern recognition techniques.
Our invention has several advantages over methods referred to in WO 92/19975. First, in our invention, no separation steps are involved which reduces the time, labor, cost and complexity of the assay. Second, no recognition elements are used in the methods of WO 92/19975, limiting the flexibility of the assay. Third, no recognition elements are used in the methods of WO 92/19975, thus, the analysis of known or unknown binding functions is not possible. And finally, the WO 92/19975 assay cannot be expanded which restricts the ability of the assay to take full advantage of pattern recognition programs.